In an attempt to better understand the molecular mechanism by which ATP is formed in mammalian tissue, the molecular mechanism by which the terminal high energy bond of ATP is made available for energy-requiring processes in both mitochondrial and extramitochondrial compartments, and the mechanism by which these processes are regulated, this proposal will focus on a continuation of our studies on three interrelated problems. The first problem is a study of the mitochondrial ATPase of rat liver as it exists in a water-soluble, oligomycin-insensitive form (Catterall and Pedersen, JBC, 246, 4987, 1971), and as it exists as a detergent-soluble, oligomycin-sensitive complex (See Progress Report). Specifically, experiments will be conducted to determine and compare the structural, catalytic, conformational, and substrate-product binding properties of these two forms of mitochondrial ATPase. Such studies should be essential to a complete understanding of the mechanism of the terminal enzymatic steps of oxidative phosphorylation and the role of the multiple ATPase subunits (Catterall and Pedersen, JBC, In Press, 1973) in this process. The second problem is a study of the uncoupler-sensitive ATPase activity of mitochondria from normal and neoplastic tissue. Since mitochondria from hepatoma and leukemic cells are markedly deficient in this activity (Pedersen et al., PNAS, 68, 1079, 1971, and Progress Report), an attempt will be made to purify the ATPase from one of these mitochondrial types and compare its structural and catalytic properties with the ATPase of nonmalignant tissue. In addition, experiments will be designed to determine the generality of this defect in both hydrocarbon and virally induced tumors. The third problem is a continuation of our studies aimed at elucidating the role of mitochondrial nucleoside diphosphokinases (Pedersen, JBC, 248, 3956, 1973). Specifically, experiments will be designed to determine the number of different species in rat liver mitochondria, their intramitochondrial localization, and their kinetic and physical distinctions.